The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3982-3991 ◽  
Author(s):  
Ana Sauri ◽  
Zora Soprova ◽  
David Wickström ◽  
Jan-Willem de Gier ◽  
Roel C. Van der Schors ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Critical Role ◽  
Structural Data ◽  
Intermediate Stage ◽  
Gram Negative Bacteria ◽  
Passenger Domain ◽  
Gram Negative ◽  
Periplasmic Chaperone ◽  
Folded Structures

Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms a β-barrel pore in the outer membrane implicated in translocation of the upstream ‘passenger’ domain across the outer membrane. However, recent structural data suggest that the diameter of the β-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage the β-domain was not inserted and folded as an integral β-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Bam (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane.

scholarly journals Bacterial Outer Membrane Proteins Are Targeted to the Bam Complex by Two Parallel Mechanisms

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Xu Wang ◽  
Janine H. Peterson ◽  
Harris D. Bernstein
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Sequence Motif ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Conserved Sequence ◽  
Bam Complex ◽  
Exact Function ◽  
Periplasmic Chaperone

ABSTRACT Membrane proteins that are integrated into the outer membrane of Gram-negative bacteria typically contain a unique “β barrel” structure that serves as a membrane spanning segment. A conserved “β signal” motif is located at the C terminus of the β barrel of many outer membrane proteins (OMPs), but the function of this sequence is unclear. We found that mutations in the β signal slightly delayed the assembly of three model Escherichia coli OMPs by reducing their affinity for the barrel assembly machinery (Bam) complex, a heterooligomer that catalyzes β barrel insertion, and led to the degradation of a fraction of the protein in the periplasm. Interestingly, the absence of the periplasmic chaperone SurA amplified the effect of the mutations and caused the complete degradation of the mutant proteins. In contrast, the absence of another periplasmic chaperone (Skp) suppressed the effect of the mutations and considerably enhanced the efficiency of assembly. Our results reveal the existence of two parallel OMP targeting mechanisms that rely on a cis-acting peptide (the β signal) and a trans-acting factor (SurA), respectively. Our results also challenge the long-standing view that periplasmic chaperones are redundant and provide evidence that they have specialized functions. IMPORTANCE Proteins that are embedded in the outer membrane of Gram-negative bacteria (OMPs) play an important role in protecting the cell from harmful chemicals. OMPs share a common architecture and often contain a conserved sequence motif (β motif) of unknown function. Although OMPs are escorted to the outer membrane by proteins called chaperones, the exact function of the chaperones is also unclear. Here, we show that the β motif and the chaperone SurA both target OMPs to the β barrel insertion machinery in the outer membrane. In contrast, the chaperone Skp delivers unintegrated OMPs to protein degradation complexes. Our results challenge the long-standing view that chaperones are functionally redundant and strongly suggest that they have specialized roles in OMP targeting and quality control.

scholarly journals The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

2021 ◽  
Vol 119 (1) ◽  
pp. e2114997119
Author(s):  
Ashton N. Combs ◽  
Thomas J. Silhavy
Keyword(s):  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Adaptor Protein ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Timely Manner ◽  
Gram Negative ◽  
Bam Complex ◽  
Periplasmic Chaperone ◽  
Chaperone Network

The biogenesis of integral β-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the β-barrel assembly machine (Bam) complex. We find that β-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam–OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

scholarly journals The gain-of-function allelebamAE470Kbypasses the essential requirement for BamD in β-barrel outer membrane protein assembly

2020 ◽  
Vol 117 (31) ◽  
pp. 18737-18743 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Meera Gupta ◽  
Martin Wühr ◽  
Thomas J. Silhavy
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Protein Assembly ◽  
Gram Negative Bacteria ◽  
Gain Of Function ◽  
Essential Requirement ◽  
Gram Negative ◽  
Selective Permeability ◽  
Catalytic Role ◽  
Global Defect

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral β-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the β-barrel assembly machine (BAM), which is composed of one OMP—BamA—and four lipoproteins—BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation,bamAE470K, that bypasses the requirement for BamD. AlthoughbamD::kanbamAE470Kcells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.

scholarly journals Functions of the BamBCDE Lipoproteins Revealed by Bypass Mutations in BamA

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Elizabeth M. Hart ◽  
Thomas J. Silhavy
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Bam Complex ◽  
Essential Function ◽  
The Individual

ABSTRACT The heteropentomeric β-barrel assembly machine (BAM complex) is responsible for folding and inserting a diverse array of β-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. The BAM complex contains two essential proteins, the β-barrel OMP BamA and a lipoprotein BamD, whereas the auxiliary lipoproteins BamBCE are individually nonessential. Here, we identify and characterize three bamA mutations, the E-to-K change at position 470 (bamAE470K), the A-to-P change at position 496 (bamAA496P), and the A-to-S change at position 499 (bamAA499S), that suppress the otherwise lethal ΔbamD, ΔbamB ΔbamC ΔbamE, and ΔbamC ΔbamD ΔbamE mutations. The viability of cells lacking different combinations of BAM complex lipoproteins provides the opportunity to examine the role of the individual proteins in OMP assembly. Results show that, in wild-type cells, BamBCE share a redundant function; at least one of these lipoproteins must be present to allow BamD to coordinate productively with BamA. Besides BamA regulation, BamD shares an additional essential function that is redundant with a second function of BamB. Remarkably, bamAE470K suppresses both, allowing the construction of a BAM complex composed solely of BamAE470K that is able to assemble OMPs in the absence of BamBCDE. This work demonstrates that the BAM complex lipoproteins do not participate in the catalytic folding of OMP substrates but rather function to increase the efficiency of the assembly process by coordinating and regulating the assembly of diverse OMP substrates. IMPORTANCE The folding and insertion of β-barrel outer membrane proteins (OMPs) are conserved processes in mitochondria, chloroplasts, and Gram-negative bacteria. In Gram-negative bacteria, OMPs are assembled into the outer membrane (OM) by the heteropentomeric β-barrel assembly machine (BAM complex). In this study, we probe the function of the individual BAM proteins and how they coordinate assembly of a diverse family of OMPs. Furthermore, we identify a gain-of-function bamA mutant capable of assembling OMPs independently of all four other BAM proteins. This work advances our understanding of OMP assembly and sheds light on how this process is distinct in Gram-negative bacteria.

scholarly journals TP0453, a Concealed Outer Membrane Protein of Treponema pallidum, Enhances Membrane Permeability

2005 ◽  
Vol 187 (18) ◽  
pp. 6499-6508 ◽  
Author(s):  
Karsten R. O. Hazlett ◽  
David L. Cox ◽  
Marc Decaffmeyer ◽  
Michael P. Bennett ◽  
Daniel C. Desrosiers ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Nutrient Transport ◽  
Treponema Pallidum ◽  
Protein S ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Membrane Spanning

ABSTRACT The outer membrane of Treponema pallidum, the noncultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning β-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive β-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic α-helices. Insertion of the recombinant, nonlipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.

scholarly journals Antiproliferative effects of porins extracted from Klebsiella pneumoniae on HL-60, NIH/3T3 and Human foreskin fibroblast cell (HFFC) lines measured by ELISA method

2010 ◽  
Vol 4 (1) ◽  
pp. 28-35
Author(s):  
Amir H. Al–Shammary ◽  
Essam F. Al-Jumaily ◽  
Nidhal Abdulmohymen
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Primary Source ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Gram Negative ◽  
Isolation And Purification ◽  
Antiproliferative Effects

Approximately, 50% of the dry mass of the outer membrane of gram-negative bacteria consists of proteins, and more than 20 immunochemically distinct proteins (termed outer membrane proteins [OMPs]) have been identified. An identified local strain of Klebsiella pneumoniae was used as a primary source for the isolation and purification of porins. Multiple concentrations of purified porins (5, 10, 15, 20, 25) g/ml were incubated with three different cell lines for (24, 72 , 120) hrs, after the end of the incubation periods, the cells were treated with Cell proliferation ELISA, BrdU (colorimetric) kit to evaluate the antiproliferative effects of porins. The results revealed that porins are potent antiproliferative agent in a time and concentration dependent manner and thus could greatly affect prokaryote-eukaryote interaction as well as the whole inflammatory process resulted after infection with gram negative bacteria.

scholarly journals Gram-negative outer-membrane proteins with multiple β-barrel domains

2021 ◽  
Vol 118 (31) ◽  
pp. e2104059118
Author(s):  
Ron Solan ◽  
Joana Pereira ◽  
Andrei N. Lupas ◽  
Rachel Kolodny ◽  
Nir Ben-Tal
Keyword(s):  
Outer Membrane ◽  
De Novo ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Single Chain ◽  
Gram Negative ◽  
Complementary Function ◽  
Beta Barrels ◽  
Β Sheets ◽  
The Individual

Outer-membrane beta barrels (OMBBs) are found in the outer membrane of gram-negative bacteria and eukaryotic organelles. OMBBs fold as antiparallel β-sheets that close onto themselves, forming pores that traverse the membrane. Currently known structures include only one barrel, of 8 to 36 strands, per chain. The lack of multi-OMBB chains is surprising, as most OMBBs form oligomers, and some function only in this state. Using a combination of sensitive sequence comparison methods and coevolutionary analysis tools, we identify many proteins combining multiple beta barrels within a single chain; combinations that include eight-stranded barrels prevail. These multibarrels seem to be the result of independent, lineage-specific fusion and amplification events. The absence of multibarrels that are universally conserved in bacteria with an outer membrane, coupled with their frequent de novo genesis, suggests that their functions are not essential but rather beneficial in specific environments. Adjacent barrels of complementary function within the same chain may allow for functions beyond those of the individual barrels.

scholarly journals The essential inner membrane protein YejM is a metalloenzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Uma Gabale ◽  
Perla Arianna Peña Palomino ◽  
HyunAh Kim ◽  
Wenya Chen ◽  
Susanne Ressl
Keyword(s):  
Antibiotic Resistance ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Critical Role ◽  
Membrane Properties ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Periplasmic Domain ◽  
Inner Membrane Protein

Abstract Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

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